title: Characterization of norovirus RNA replicase for in vitro amplification of RNA.
abstract: BACKGROUND
  The isothermal amplification of RNA in vitro has been used for the study of in vitro evolution of RNA. Although Qβ replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3D(pol)) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3D(pol) in vitro were re-evaluated in this context.
  
  
  RESULTS
  NV3D(pol), synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3'-terminal structure of single-stranded RNA template, and especially, NV3D(pol) preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3'-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3D(pol) also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3D(pol) in vitro.
  
  
  CONCLUSIONS
  NV3D(pol) can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences.
authors.affiliation: Innovative Research Organization, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama 338-8570, Japan. husimi@mail.saitama-u.ac.jp.
authors.forename: Hidenao
authors.initials: H
authors.lastname: Arai
authors.forename: Koichi
authors.initials: K
authors.lastname: Nishigaki
authors.forename: Naoto
authors.initials: N
authors.lastname: Nemoto
authors.forename: Miho
authors.initials: M
authors.lastname: Suzuki
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D029322:Norovirus / Q000201:enzymology*; D021141:Nucleic Acid Amplification Techniques; D009690:Nucleic Acid Conformation; D011777:Q beta Replicase / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000378:metabolism
chemical_list: C034477:RNA primers; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012313:RNA; D011777:Q beta Replicase; D012324:RNA-Dependent RNA Polymerase